GSI-I (Z-LLNle-CHO) inhibits γ-secretase and the proteosome to trigger cell death in precursor-B acute lymphoblastic leukemia

Gamma secretase inhibitors (GSIs) comprise a growing class of compounds that interfere with the membrane-bound Notch signaling protein and its downstream intra-nuclear transcriptional targets. As GSI-I (Z-LLNle-CHO) is also a derivative of a widely used proteosome inhibitor MG-132, we hypothesized that this compound might be active in precursor-B acute lymphoblastic leukemia (ALL) cell lines and patient samples. We found that GSI-I treatment of precursor-B ALL blasts induced apoptotic cell death within 18–24 h. With confirmation using RNA and protein analyses, GSI-I blocked nuclear accumulation of cleaved Notch1 and Notch2, and inhibited Notch targets Hey2 and Myc. Microarray analyses of 207 children with high-risk precursor-B ALL demonstrate that Notch pathway expression is a common feature of these neoplasms. However, microarray studies also implicated additional transcriptional targets in GSI-I-dependent cell death, including genes in the unfolded protein response, nuclear factor-κB and p53 pathways. Z-LLNle-CHO blocks both γ-secretase and proteosome activity, inducing more robust cell death in precursor-B ALL cells than either proteosome-selective or γ-secretase-selective inhibitors alone. Using Z-LLNle-CHO in a nonobese diabetes/severe combined immunodeficiency (NOD/SCID) precursor-B ALL xenograft model, we found that GSI-I alone delayed or prevented engraftment of B-lymphoblasts in 50% of the animals comprising the experimental group, suggesting that this compound is worthy of additional testing

Reprogramming of myeloid progenitors by the BCR-ABL oncogene

The constitutive tyrosine kinase activity of the BCR-ABL oncogene is central for the pathogenesis of chronic myeloid leukaemia (CML). Clinical treatment of CML with the kinase inhibitor Imatinib Mesylate (IM) is a fundamental success demonstrating disease addiction upon BCR-ABL activity. However, 15-20% of CML patients develop drug resistance leading to patient fatality. The most studied mechanism of IM-resistance is point mutations within the kinase domain of BCR-ABL. However, these account for ~40% of patients crafting the challenge in understanding how leukaemic cells can survive in the absence of BCR- ABL activity. To address this issue, a preclinical model of CML drug resistance was established using KCL22 cells (Chapter 3). Single cell clones were generated and drug resistant derivatives established which had (i) no mutations within BCR-ABL and (ii) functional IM pharmacokinetics with reduction in the phosphorylation of STAT5 and CrkL. Being kinase-independent, are other domains of BCR-ABL compensating to maintain the leukaemic state or is BCR-ABL now redundant? To address this, the consequence of BCR-ABL depletion was explored (Chapter 4). In contrast to the original parental KCL22 cells, the drug-resistant clones continued to survive and expand upon the loss of BCR-ABL, supporting the proposal of ‘oncogene independence’ as a novel mechanism to escape drug targeting. Genome-wide expression analysis identified the induction of the RUNX1 transcription factor upon drug resistance. As RUNX1 is deregulated is several haematological malignancies, its potential role in drug resistant CML was explored (Chapter 5). Targeting of RUNX1 within drug resistant cells resulted in the failure of these cells to grow associated with the acquisition of a macrophage-like phenotype. The work herein identifies oncogene independence as a novel mechanism for establishing drug resistant CML with RUNX1 functioning as new oncogenic driver to compensate for the loss of BCR-ABL and presenting itself as a new therapeutic target

Image1_Sirtuin1 meditated modification of Notch1 intracellular domain regulates nucleolar localization and activation of distinct signaling cascades.pdf

Notch signaling is involved in cell fate decisions in the development and maintenance of tissue homeostasis. Spatial regulation of the Notch1 intracellular domain (NIC1), has been shown to underpin signaling outcomes mediated by this receptor. We recently reported a putative Nucleolar Localization Sequence (NoLS) in NIC1. Here we investigate if the putative NoLS identified in NIC1 regulates localization in the nucleolus and anti-apoptotic activity. Confocal imaging of live cells expressing NIC1 or forms modified by deletion or site-directed mutagenesis established that the putative NoLS in NIC1 is required for nucleolar localization and regulated by the deacetylase Sirtuin1. Subsequent analysis of anti-apoptotic activity revealed signaling cascades linked to nucleolar localization. For this, etoposide and 4-Nitroquinoline 1-oxide, an inhibitor of topoisomerase-II and a UV mimetic drug respectively, were used as prototypic triggers of genomic damage in a mammalian cell line. While NIC1 blocked apoptosis regardless of its localization to the nucleoplasm or nucleolus, modifications of NIC1 which promoted localization to the nucleolus triggered a dependence on the nucleolar proteins fibrillarin and nucleolin for anti-apoptotic activity. Further, cells co-expressing NIC1 and Sirtuin1 (but not its catalytically inactive form), confirmed both spatial regulation and the switch to dependence on the nucleolar proteins. Finally, site-directed mutagenesis showed that the NoLS lysine residues are targets of Sirtuin1 activity. NIC1 mediated transcription is not similarly regulated. Thus, NIC1 localization to the nucleolus is regulated by Sirtuin1 modification of the lysine residues in NoLS and triggers a distinct signaling cascade involving nucleolar intermediates for anti-apoptotic activity.</p

Evidence for a role for notch signaling in the cytokine-dependent survival of activated T cells

Peripheral T cell homeostasis results from a balance between factors promoting survival and those that trigger deletion of Ag-reactive cells. The cytokine IL-2 promotes T cell survival whereas reactive oxygen species (ROS) sensitize T cells to apoptosis. Two pathways of activated T cell apoptosis-one triggered by Fas ligand and the other by cytokine deprivation-depend on ROS, with the latter also regulated by members of the Bcl-2 family. Notch family proteins regulate several cell-fate decisions in metazoans. Ectopic expression of the Notch1 intracellular domain (NICD) in T cells inhibits Fas-induced apoptosis. The underlying mechanism is not known and the role, if any, of Notch in regulating apoptosis triggered by cytokine deprivation or neglect has not been examined. In this study, we use a Notch1/Fc chimera; a blocking Ab to Notch1 and chemical inhibitors of γ -secretase to investigate the role of Notch signaling in activated T cells of murine origin. We show that perturbing Notch signaling in activated CD4+/CD8+ T cells maintained in IL-2 results in the accumulation of ROS, reduced Akt/protein kinase B activity, and expression of the antiapoptotic protein Bcl-xL, culminating in apoptosis. A broad-spectrum redox scavenger inhibits apoptosis but T cells expressing mutant Fas ligand are sensitive to apoptosis. Activated T cells isolated on the basis of Notch expression (Notch+) are enriched for Bcl-xL expression and demonstrate reduced susceptibility to apoptosis triggered by neglect or oxidative stress. Furthermore, enforced expression of NICD protects activated T cells from apoptosis triggered by cytokine deprivation. Taken together, these data implicate Notch1 signaling in the cytokine-dependent survival of activated T cells

Evidence for a Role for Notch Signaling in the Cytokine-Dependent Survival of Activated T Cells

Peripheral T cell homeostasis results from a balance between factors promoting survival and those that trigger deletion of Ag-reactive cells. The cytokine IL-2 promotes T cell survival whereas reactive oxygen species (ROS) sensitize T cells to apoptosis. Two pathways of activated T cell apoptosis–one triggered by Fas ligand and the other by cytokine deprivation–depend on ROS, with the latter also regulated by members of the Bcl-2 family. Notch family proteins regulate several cell-fate decisions in metazoans. Ectopic expression of the Notch1 intracellular domain (NICD) in T cells inhibits Fas-induced apoptosis. The underlying mechanism is not known and the role, if any, of Notch in regulating apoptosis triggered by cytokine deprivation or neglect has not been examined. In this study, we use a Notch1/Fc chimera; a blocking Ab to Notch1 and chemical inhibitors of $\gamma$-secretase to investigate the role of Notch signaling in activated T cells of murine origin. We show that perturbing Notch signaling in activated $CD4^+/CD8 ^+$ T cells maintained in IL-2 results in the accumulation of ROS, reduced Akt/protein kinase B activity, and expression of the antiapoptotic protein $Bcl-xL$, culminating in apoptosis. A broad-spectrum redox scavenger inhibits apoptosis but T cells expressing mutant Fas ligand are sensitive to apoptosis. Activated T cells isolated on the basis of Notch expression $(Notch^+)$ are enriched for $Bcl-xL$ expression and demonstrate reduced susceptibility to apoptosis triggered by neglect or oxidative stress. Furthermore, enforced expression of NICD protects activated T cells from apoptosis triggered by cytokine deprivation. Taken together, these data implicate Notch1 signaling in the cytokine-dependent survival of activated T cells

Notch1 is an effector of Akt and hypoxia in melanoma development

Melanomas are highly aggressive neoplasms resistant to most conventional therapies. These tumors result from the interaction of altered intracellular tumor suppressors and oncogenes with the microenvironment in which these changes occur. We previously demonstrated that physiologic skin hypoxia contributes to melanomagenesis in conjunction with Akt activation. Here we show that Notch1 signaling is elevated in human melanoma samples and cell lines and is required for Akt and hypoxia to transform melanocytes in vitro. Notch1 facilitated melanoma development in a xenograft model by maintaining cell proliferation and by protecting cells from stress-induced cell death. Hyperactivated PI3K/Akt signaling led to upregulation of Notch1 through NF-κB activity, while the low oxygen content normally found in skin increased mRNA and protein levels of Notch1 via stabilization of HIF-1α. Taken together, these findings demonstrate that Notch1 is a key effector of both Akt and hypoxia in melanoma development and identify the Notch signaling pathway as a potential therapeutic target in melanoma treatment